The basic helix-loop-helix (bHLH) superfamily of transcription factors regulates growth and differentiation in a variety of tissues by forming transcriptionally active heterodimers that bind to E-box (CANNTG) elements in the promoters of target genes. Recently, we have obtained indirect evidence for involvement of the bHLH factors in the regulation of osteocalcin gene expression in osteoblasts. Our preliminary studies have identified putative bHLH proteins in mature osteoblasts that bind to cis- acting E-box elements in the osteocalcin promoter and regulate osteocalcin gene transcription. In the proposed investigations, we will further characterize these bHLH transcription factors and define their role in regulating osteoblast development. In initial studies we will employ adenoviral vectors to introduce the negative regulatory HLH protein, Id1, as well as osteocalcin promoter-reporter constructs into Ros 17/2.8 and post-mitotic MC3T3-E1 osteoblast models. These studies will permit examination of Id forced expression on osteocalcin gene transcription and E-box binding activity under basal and hormonally stimulated conditions. We will also determine if bHLH proteins play a broader role in regulating the osteoblast developmental program. To accomplish this, we will stably express Id in the MC3T3-E1 cells using retroviral vectors and examine its effects on the temporal sequence of osteoblast development. Next, we will evaluate the function of cis-acting elements responsible for Id inhibition of osteocalcin gene expression by deletional and mutational analysis of the three E-box binding sites located in the osteocalcin promoter. Finally, to identify and eventually clone potentially novel members of the bHLH protein family involved in osteoblast growth and development, we will use complementary strategies that include screening an osteoblast expression library with E-box oligonucleotide probes and/or screening an osteoblast cDNA library with cDNA probes derived from conserved regions of bHLH proteins that are differentially displayed during osteoblast development. Our investigations will add significant information to our understanding of genes with critical roles in regulating osteoblast growth and development. Such knowledge may contribute to elucidating the pathogenesis of and defining novel pharmacologic ways to treat osteopenic disorders.